Lu Liqun, Kwang Jimmy
Animal Health Biotechnology Unit, Temasek Life Science Laboratory, National University of Singapore, Singapore 117604, Singapore.
FEBS Lett. 2004 Nov 5;577(1-2):141-6. doi: 10.1016/j.febslet.2004.08.087.
To characterize the role of latency-associated ORF427 of white spot syndrome virus (WSSV), a shrimp cDNA library was constructed to screen interacting proteins of ORF427. Employing the yeast two-hybrid system, a novel shrimp protein phosphatase (named PPs), sharing 93% homology with human protein phosphatase 1, has been identified able to bind ORF427 in yeast. Through co-immunoprecipitation assays, the interaction between PPs and ORF427 was further confirmed both in vitro and in vivo. Interestingly, the novel shrimp protein phosphatase consists of only 199 aa and contains almost all the functional catalytic domains of human protein phosphatase, while it lacks the corresponding C-terminal non-catalytic sequence. Transcription and translation products of the identified cDNA can be detected in both normal and WSSV-infected shrimps; and PPs was found to localize mainly in the lysosome of shrimp cells. To characterize its function, the PPs cDNA was highly expressed in bacteria and the purified protein showed phosphatase activity when tested against pNPP in a standard phosphatase assay. Our results suggest that the identified protein phosphatase, PPs, may represent a novel member of protein phosphatase family and might be involved in the regulation of WSSV's life cycle through interaction with latency-related ORF427 of WSSV.
为了表征白斑综合征病毒（WSSV）潜伏期相关开放阅读框427的作用，构建了一个虾cDNA文库以筛选与开放阅读框427相互作用的蛋白质。利用酵母双杂交系统，已鉴定出一种与人类蛋白磷酸酶1具有93%同源性的新型虾蛋白磷酸酶（命名为PPs），它能够在酵母中与开放阅读框427结合。通过免疫共沉淀试验，进一步在体外和体内证实了PPs与开放阅读框427之间的相互作用。有趣的是，这种新型虾蛋白磷酸酶仅由199个氨基酸组成，几乎包含了人类蛋白磷酸酶的所有功能催化结构域，而缺少相应的C端非催化序列。在正常虾和感染WSSV的虾中均能检测到所鉴定cDNA的转录和翻译产物；并且发现PPs主要定位于虾细胞的溶酶体中。为了表征其功能，PPs cDNA在细菌中高度表达，纯化后的蛋白在标准磷酸酶检测中以对硝基苯磷酸酯（pNPP）为底物进行检测时显示出磷酸酶活性。我们的结果表明，所鉴定的蛋白磷酸酶PPs可能代表蛋白磷酸酶家族的一个新成员，并且可能通过与WSSV潜伏期相关的开放阅读框427相互作用参与WSSV生命周期的调控。
